| Background:Activation of transforming growth factor-β1 (TGF-β1)-Smad3 pathway aggravates myocardial ischemia/reperfusion injury (IRI). TGF-(31-Smad3 pathway is activated in diabetic heart, which is one possible reason for the worse prognosis after myocardial IRI in the patients with diabetes. We previously showed that glutamine (Gin) protects cardiomyocytes from hypoxia/reoxygenation (H/R) injury under high glucose (HG) conditions. The aim of this study was to investigate whether Gin exerts its protective effect in IRI via inhibiting TGF-β1-Smad3 pathway.Methods:In vitro, H9c2 rat cardiomyocytes were treated with HG (33 mM) and/or H/R. Firstly, TGF-β1 receptor inhibitor (SB431542), Smad3 inhibitor (SIS3) were used to inhibit the activation of Smad3 pathway to observe the status of the cells. Secondly, different concentrations of Gin were used to proctect cells from injury and the changes of the TGF-β1-Smad3 pathway were detected. At last, TGF-β1 was used to stimulate Smad3 pathway to further test the hypothesis. We also performed in vivo experiments in which we treated normal and diabetic rats with Gin or solvent control following IRI. We assessed protein levels of TGF-β1, total Smad3, phosphorylated (p)-Smad3 and cleaved caspase-3 in H9c2 cells and rat myocardium by Western blotting. We test the apoptosis rate of cardiomyocytes and heart tissue by TUNEL assay. We also measured myocardial infarction size, myocardial microstructure and hemodynamics of rats.Results:In vitro, H9c2 cells treated with HG+H/R exhibited high apoptosis rates, as well as a highly activated TGF-β1-Smad3 pathway. TGF-β1 receptor inhibitor (SB431542) or Smad3 inhibitor (SIS3) reduced HG+H/R induced apoptosis. Similarly, Gin supplementation alleviated apoptosis and decreased p-Smad3 levels. However, Gin’s protective effect was significantly weakened by TGF-β1. In vivo, diabetic rats without treatment exert severe myocardial injury after IRI. However, diabetic rats treated with Gin had improved hemodynamics, smaller infarct size, lower rate of apoptosis, lighter structural damage after IRI, and a significant decrease in TGF-β1-Smad3 pathway activation.Conclusions:Thus, we conclude that the activation of TGF-β1-Smad3 pathway aggravates IRI in diabetic heart; Gln decreases IRI of diabetic heart partly through inhibiting the activation of the TGF-β1-Smad3 pathway.Background:In the first part of the study, we established a model of myocardial ischemia and reperfusion injury (IRI) in SD rats. The heart size and structure of small rodents are much different from the human heart, so these animals are limited in some experimental applications. The large animals have unique advantages. Such as the beagles, they have similar heart structure, physiopathologic reaction with human and have developed circulatory system, which is more suitable for the study of cardiovascular disease. Furthermore, their genetic performance are stable and excellent. At present, there is no animal models can be used for evaluating the efficacy of new thrombolytic agents with similar clinical status. So in the second part of the study, at first, we analyzed the composition of clinical coronary thrombus, then inject self-made embolus into coronary artery by minimally invasive interventional technique to establish a beagle model for evaluating the efficacy of thrombolytic drugs.This model also can be used to study the mechanism of IRI after thrombolysis.Methods:18 male beagles were included and divided into three groups:red embolus group (n=6), white embolus group (n=6) or white embolus+rt-PA group (n=6). Autologous emboli were infused into the mid-distal region of the left anterior descending coronary artery. Electrocardiogram was used to monitor the progresses of coronary artery embolism and reperfusion. Coronary angiography was performed to verify the status of embolism. The composition of embolus was examined by HE staining and scanning electron microscope (SEM). Myocardial infarct size was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining.Results:Red thrombus was characteristic of loose reticular structure of erythrocytes under HE staining and SEM, while the white embolus had compacted structure that mainly consisted of a dense mass of fibrin. Coronary angiography showed the recanalization rate was 2/6 in the red embolus group versus 0/6 in the white embolus group in three hours after occlusion. Arrhythmia, resolution of ST-segment elevation and lower T wave on the electrocardiogram appeared in the red embolus group but not in the white embolus group. Another six dogs with white thrombi were treated with rt-PA. Five out of six dogs exhibited coronary recanalization after two hours of therapy, compared to zero dogs without rt-PA treatment. And the recanalization time was 43.2±7.4 minutes in rt-PA group. The size of myocardial infarction in rt-PA group reduced significantly compared with white embolus group using TTC staining method.Conclusions:The white embolism model was more convenient experimentally and had a higher uniformity, stability and success rate. The major innovation of our study is that we applied fibrin-rich white thrombi to establish beagle model possessing features of clinically observed coronary thrombi in time window of intravenous thrombolysis of ST-elevation myocardial infarction (STEMI). This model can be used to evaluate new thrombolytic drugs for the treatment of STEMI. The model simulates the state of myocardial IRI after thrombolysis, and it can be used to investigate the pathological changes and mechanism of myocardial IRI after thrombolysis. |
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